Fluorescence microscopy is widely used in the biological sciences to study the three-dimensional interior of cells and organisms and to visualize particular biomolecules with specificity through fluorescent labeling. However, one of fluorescence microscopy's greatest weaknesses is moderate spatial resolution, which is fundamentally limited by the wavelength of light. The typical wide-field light microscope has another weakness in that it does not gather sufficient complete information about a sample to allow true three-dimensional imaging, which is called the missing cone problem. The manifestation of the missing cone problem in the raw image data, which are acquired as a sequence of two-dimensional images, often referred to as sections with different focus, is that each section of the data contains not only in-focus information from the corresponding section of the sample but also out-of-focus blur from all other sections. Three-dimensional reconstructions from conventional microscope data presently have to rely on a priori constraints, such as the nonnegativity of the density of fluorescent dye, to attempt to compensate for the missing information.
Confocal microscopy is one technique that addresses both weaknesses by using a pinhole aperture to physically block the out-of-focus light from reaching the detector. Confocal microscopy provides true three-dimensional imaging and at the same time extends resolution somewhat beyond the conventional limit, both axially (i.e., depth) and laterally. The improvement of lateral resolution, however, only takes place when a small pinhole is used, but by using a small pinhole much of the in-focus light is discarded along with the unwanted out-of-focus light. In practice, it is rarely advantageous to use such a small pinhole given the weak fluorescence of typical biological samples and the low sensitivity of detectors normally used in confocal microscopes. The detrimental loss of in-focus light usually outweighs any resolution benefits. As a result, typical confocal microscopes are operated with wider pinholes, producing a lateral resolution that is only a marginal improvement over that provided by conventional wide-field fluorescence microscopes.
In recent years, it has been demonstrated that it is possible to double the lateral resolution of the fluorescence microscope without significant loss of light using spatially structured illumination light to frequency-mix high resolution information into the optical passband of the microscope. In particular, three-dimensional structured illumination microscopy (“3D-SIM”) achieves improvement in lateral and axial resolution by a factor of two when compared to confocal microscopy. Because 3D-SIM requires no specialized fluorescent dyes or proteins, biologists achieve high resolution with 3D-SIM and retain convenient and familiar fluorescence labeling techniques. Multiple images of the subject are made by moving three-dimensional structured illumination pattern through the sample. Higher resolution is achieved by solving a system of equations to restore the fine spatial detail normally blurred by diffraction. Even with the improvements in lateral and axial resolution offered by 3D-SIM, scientists, engineers, and microscope manufactures continue to seek data processing methods and microscopy systems that increase the lateral and axial resolution of sample components using fluorescence microscopes.